Detecting and characterizing the thermodynamics of a chemical reaction provides insight into the mechanism by which the chemical reaction occurs. For example, the detection and characterization of binding interactions (e.g., protein-protein interactions, proteins-DNA, drug-protein interactions) are central to basic biological research and pharmaceutical research and development (R&D). Many, analytical techniques have been developed to study various types and aspects of binding interactions. Some examples of these techniques include Enzyme-Linked ImmunoSorbent Assay (ELISA), mass spectrometry, fluorescence resonance energy transfer, fluorescence correlation spectroscopy, fluorescence anisotropy, protein arrays, nucleic acid microarrays, and calorimetry. Calorimetry is a particularly advantageous method used to study the thermodynamics of binding interactions. Calorimetry measures the energy released or absorbed by a reaction over a range of reactant concentrations, and uses this information to determine the thermodynamic properties, stoichiometry, and equilibrium binding constant for the reaction.
In a typical calorimetry experiment, the heat of reaction, enthalpy (ΔH), is measured and from this measurement the Gibbs free energy, entropy, affinity constant, and stoichiometry are determined. A reaction in which heat is released is exothermic (positive ΔH), whereas a reaction that absorbs heat is endothermic (negative ΔH). An accurate measurement of enthalpy and subsequent determination of entropy allows for an assessment of the relative contributions of each to the binding interactions of particular compounds. Understanding these relative contributions allows for the selection of compounds that are more readily optimized for various applications (e.g., pharmaceutical development).
FIG. 1 generally outlines a calorimetry process and exemplary information obtained from the process. In FIG. 1, a reaction of interest 50 comprises a drug candidate 52 and a target protein 54. By measuring the heat 56 released or absorbed by the reaction, information 58 may be derived from the heat signature of the reaction 50, and the information 58 may be used, for example, to help determine the potential efficacy of the drug candidate. The information 58 may include indicators of bioactivity, such as the number of bonds formed, type of bonds, physical fit of the ligand (drug candidate) in the binding site of the target protein, binding kinetics, and stoichiometry, although other information may also be included in information 58.
Enthalpy (ΔH) is driven primarily by the number and type of bonds in the binding reaction, and provides an indication of specific interactions between binding partners. Enthalpy determinations help ensure specificity, selectivity and adaptability of the binding compounds. More specifically, enthalpy corresponds to the energy associated with the net change in noncovalent bonds between binding partners. Noncovalent bonds help maintain the three-dimensional structure of large molecules such as proteins and nucleic acids, and are involved in many biological processes in which large molecules bind specifically but transiently to one another. As such, a larger ΔH suggests a better complementarity of bonds in the interface, and a comparison of ΔH's of binding reactions provides useful information for selecting between compounds having similar affinities, thereby determining which compounds should undergo further chemical modification. By comparison, entropy is driven primarily by the geometry of the binding compounds, and may play a lesser role in characterizing binding specificity.
Temperature sensors conventionally employed for determining the heat of a chemical reaction in calorimetry studies include thermocouples, thermopiles, and/or thermistors. For example, in one calorimetry study described by Torres et al. (Torres et al. Enthalpy Arrays, PNAS 101, 9517-9522, 2004), enthalpy arrays are based on amorphous silicon thermistors fabricated via photolithography with 50 micrometer design rules, where each sensor includes of two thermistors connected in a Wheatstone bridge configuration. In Torres' system, samples consisting of two small drops of liquid, one for each reactant, are electrostatically merged, and the difference between the temperatures of the respective sensors indicates the heat of the reaction.
Other temperature sensing methods have used changes in optical properties to infer temperature changes in reactions with immobilized reactants (e.g., Zhang et al., Calorimetric biosensors with integrated microfluidic channels. Biosensors and Bioelectronics, 19, 1733-1743, 2004). However, these optical-based temperature-sensing approaches often lack a desired sensitivity for detecting small changes in temperature that typically characterize chemical reactions with small enthalpy changes.